Optical traps have been widely used in a large variety of applications ranging from biophysics to nano-sciences. More than one microscopic object can be captured in an optical trap. In the practical application, it is always necessary to distinguish and control the number of captured objects in the optical trap. In this paper, a novel method has been presented to distinguish the number of trapped microspheres by measuring the intensity of back signal. Clear descent of the back signal has been observed when a microsphere is captured in the center of optical trap. The relative coupling efficiency of back signal decreases as the number of captured microspheres increases both in experiment and theory. This method contributes to miniaturization and integration of applied systems due to getting rid of the imaging system, and is generally applicable to the area of nanoparticle trapping. © 2017 SPIE.